Bacterial live vector vaccines expressing chromosomally-integrated foreign antigens

ABSTRACT

Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. In the present invention, genes encoding protective antigens of unrelated bacterial, viral, parasitic, or fungal pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting relevant immune responses. Rather than expressing these antigens using only low copy expression plasmids, expression of foreign proteins is accomplished using both low copy expression plasmids in conjunction with chromosomal integrations within the same live vector. This strategy compensates for the inherent disadvantage of loss of gene dosage (versus exclusive plasmid-based expression) by integrating antigen expression cassettes into multiple chromosomal sites already inactivated in an attenuated vector.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant Number AI077911 and Grant Number AI095309 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

A sequence listing in electronic (ASCII text file) format is filed with this application and incorporated herein by reference. The name of the ASCII text file is “2016_0626_ST25.txt”; the file was created on Aug. 11, 2016; the size of the file is 92 KB.

TECHNICAL FIELD

The invention generally relates to the provision of live vector vaccines that can be used to vaccinate a subject against bacterial, viral or parasitic pathogens. In particular, the invention relates to bacterial live vector vaccines that express chromosomally-integrated antigen expression cassettes encoding selected antigens, such as protective antigens of unrelated bacterial, viral or parasitic pathogens.

BACKGROUND OF INVENTION

Excellent progress has been made over the past twenty years in the adaptation of attenuated bacterial vaccine strains for expression of foreign antigens to create multivalent live vector vaccines. This has included a devotion of significant effort to the creation of expression technologies which either directly or indirectly address the important problem of metabolic stress often associated with expression of foreign immunogens.[1,2] It is recognized that inappropriate synthesis of high levels of foreign protein in an effort to induce an antigen-specific protective immune response can adversely affect the fitness and growth rate of an already attenuated vaccine strain, resulting in over-attenuation and loss of immunity directed at both the live vector and foreign antigen. If these target immunogens are encoded by multicopy expression plasmids, these undesirable metabolic fluxes can result in plasmid loss in the absence of selective pressure, which ultimately defeats the strategy of live vector-mediated delivery of vaccine antigens.

Effective genetic stabilization systems have been developed for enhancing the retention of multicopy plasmids encoding regulated synthesis of foreign antigens, without the further requirement to select with antibiotics.[3,4,5] Antigen export systems have also been developed to reduce proteolytic degradation of foreign antigens within the cytoplasm and more effectively deliver these antigens to the immune system to enhance immunogenicity. [6,7,8,9] Thus, a variety of genetic techniques and technologies are now available for efficient delivery of one or more antigens using live vector vaccines. However, significant problems remain associated with this technology. For example inclusion of more than one gene encoding a foreign antigen of interest within a single multicopy plasmid can lead to large plasmids which ultimately prove to be genetically unstable, reducing both antigen synthesis and the ensuing immune responses.[10]

Novel strategies for engineering live vector vaccines to express high levels of a foreign antigen or to express two or more different antigens are needed.

BRIEF SUMMARY OF INVENTION

The present invention is based on a novel strategy of engineering live vector vaccines to have antigen expression cassettes encoding an antigen of interest integrated into two or more different chromosomal locations, and optionally carrying a plasmid-based expression system. Live vector vaccines engineered in this manner can deliver sufficiently immunogenic levels of the chromosomally encoded antigens to a subject. The strategic integration of antigen expression cassettes into multiple locations within the chromosome of the selected live vector results in production of sufficient levels of the encoded antigen, while avoiding adverse effects on the fitness and growth rate of the vector.

In a first embodiment, the invention is directed to an attenuated strain of Salmonella enterica serovar Typhi (hereinafter “S. Typhi”) having disruptions of two or more chromosomal locations selected from the group consisting of the guaBA locus, the htrA locus, the clyA locus, the rpoS locus, and the ssb locus. In one aspect of this embodiment, the attenuated strain of S. Typhi is the strain CVD 910 which has disruptions of the guaBA locus, the htrA locus, and the rpoS locus.

In a second embodiment, the invention is directed to an antigen-encoding attenuated strain of S. Typhi, wherein the strain comprises:

(a) disruptions of two or more chromosomal locations, wherein the chromosomal locations are selected from the group consisting of the guaBA locus, the htrA locus, the clyA locus, the rpoS locus, and the ssb locus, and

(b) chromosomal-based expression systems integrated into the locations of the chromosomal disruptions, wherein each chromosomal-based expression system comprises an antigen expression cassette encoding an antigen of interest.

In one aspect of this embodiment, the antigen of interest is a protective antigen of, for example, an unrelated bacterial, viral, parasitic, or fungal pathogen. In a particular aspect, the antigen of interest is one or more of the cell binding domain of C. difficile toxin A (CBD/A), the cell binding domain of C. difficile toxin B (CBD/B), the cell binding domain of C. difficile binary toxin (BT), the LcrV antigen of Yersinia pestis and the capsular F1 antigen of Yersinia pestis. In a further aspect, each chromosomal-based expression system comprises an antigen expression cassette encoding a different antigen of interest.

In another aspect of this embodiment, the antigen-encoding attenuated strain of S. Typhi is the strain CVD 910 which has disruptions of the guaBA locus, the htrA locus, and the rpoS locus, and which has a chromosomal-based expression system integrated into each site of disruption that encodes one or more antigens of interest. In a particular aspect, the antigen-encoding attenuated strain of S. Typhi is the strain CVD 910-3A which has disruptions of the guaBA locus, the htrA locus, and the rpoS locus, and which comprises antigen expression cassettes integrated into the locations of chromosomal disruption, wherein each antigen expression cassette encodes the cell binding domain of C. difficile toxin A.

In a third embodiment, the invention is directed to an antigen-encoding attenuated strain of S. Typhi, wherein the strain comprises:

(a) disruptions of two or more chromosomal locations, wherein the chromosomal locations are selected from the group consisting of the guaBA locus, the htrA locus, the clyA locus, the rpoS locus, and the ssb locus,

(b) chromosomal-based expression systems integrated into the locations of the chromosomal disruptions, wherein each chromosomal-based expression system comprises an antigen expression cassette encoding an antigen of interest, and

(c) one or more plasmid-based expression systems, wherein each plasmid-based expression system encodes an antigen of interest.

In one aspect of this embodiment, the antigens of interest are individually protective antigens of, for example, unrelated bacterial, viral or parasitic pathogens. In a particular aspect, the antigens of interest are individually one or more of the cell binding domain of C. difficile toxin A (CBD/A), the cell binding domain of C. difficile toxin B (CBD/B), the cell binding domain of C. difficile binary toxin (BT), the LcrV antigen of Yersinia pestis and the capsular F1 antigen of Yersinia pestis. In a further aspect, the antigens of interest are different.

In another aspect of this embodiment, the antigen-encoding attenuated strain of S. Typhi is the strain CVD 910-3A which has disruptions of the guaBA locus, the htrA locus, and the rpoS locus, and which comprises antigen expression cassettes integrated into the locations of chromosomal disruption, wherein each antigen expression cassette encodes the cell binding domain of C. difficile toxin A, a further disruption in the ssb locus, and which has an SSB-stabilized plasmid-based expression system. In a particular aspect, the antigen-encoding attenuated strain of S. Typhi is the strain CVD 910-3Assb(pSEC10-CBD/B) which has disruptions of the guaBA locus, the htrA locus, and the rpoS locus, and which comprises antigen expression cassettes integrated into the locations of chromosomal disruption, wherein each antigen expression cassette encodes the cell binding domain of C. difficile toxin A, a further chromosomal deletion of the ssb locus, and an SSB-stabilized plasmid-based expression system encoding the cell binding domain of C. difficile toxin B.

In a fourth embodiment, the invention is directed to an antigen-encoding attenuated strain of S. Typhi, wherein the strain comprises:

(a) disruptions of four chromosomal locations, wherein the chromosomal locations are selected from the group consisting of the guaBA locus, the htrA locus, the clyA locus, and the rpoS locus,

(b) chromosomal-based expression systems integrated into the locations of the chromosomal disruptions, wherein each chromosomal-based expression system comprises an antigen expression cassette encoding an antigen of interest, and

(c) a plasmid-based expression system, wherein the plasmid-based expression system encodes an antigen of interest.

The antigens of interest may be the same or different in a single strain, and a single copy or multiple copies of the same antigen can be expressed in a single strain. In one aspect of this embodiment, the antigens of interest are protective antigens of, for example, unrelated bacterial, viral or parasitic pathogens. In a particular aspect, the antigens of interest are one or more of the cell binding domain of C. difficile toxin A (CBD/A), the cell binding domain of C. difficile toxin B (CBD/B), the cell binding domain of C. difficile binary toxin (BT), the LcrV antigen of Yersinia pestis and the capsular F1 antigen of Yersinia pestis. In a further aspect, the attenuated strain of S. Typhi expresses three different antigens of interest.

In another aspect of this embodiment, the antigen-encoding attenuated strain of S. Typhi is the strain CVD 910-3A which has disruptions of the guaBA locus, the htrA locus, the rpoS locus and the clyA locus, and which comprises antigen expression cassettes integrated into the locations of chromosomal disruption, wherein at least one of the antigen expression cassettes encodes the cell binding domain of C. difficile toxin A (CBD/A), wherein at least one of the antigen expression cassettes encodes the binary toxin (BT) of C. difficile, which has a further disruption in the ssb locus, and which has an SSB-stabilized plasmid-based expression system expressing the cell binding domain of C. difficile toxin B (CBD/B). In a particular embodiment, three of the antigen expression cassettes encode the cell binding domain of C. difficile toxin A (CBD/A) and one of the antigen expression cassettes encodes the binary toxin (BT) of C. difficile. In a further particular embodiment, two of the antigen expression cassettes encode the cell binding domain of C. difficile toxin A (CBD/A) and two of the antigen expression cassettes encode the binary toxin (BT) of C. difficile.

In a particular aspect, the antigen-encoding attenuated strain of S. Typhi is the strain CVD 910-3A-GB2ssb(pSEC10-CBD/B) which comprises (i) disruptions of the guaBA locus, the htrA locus, the rpoS locus and the clyA locus, and which comprises antigen expression cassettes integrated into the locations of chromosomal disruption, wherein antigen expression cassette located in guaBA chromosomal disruption encodes the binary toxin (BT) of C. difficile, wherein antigen expression cassettes located in htrA, rpoS and clyA chromosomal disruptions encode the cell binding domain of C. difficile toxin A (CBD/A), (ii) a disruption in the ssb locus, and (iii) an SSB-stabilized plasmid-based expression system encoding the cell binding domain of C. difficile toxin B (CBD/B.

In a fifth embodiment, the invention is directed to a live vector vaccine comprising an antigen-encoding attenuated strain of S. Typhi as defined herein, and a pharmaceutically-acceptable carrier or diluent.

In a sixth embodiment, the invention is directed to methods of inducing an immune response to an antigen of interest in a subject, comprising administering to a subject an antigen-encoding live vector vaccine as defined herein that expresses an antigen of interest.

In a seventh embodiment, the invention is directed to methods of vaccinating a subject with a protective antigen, comprising administering to a subject an antigen-encoding live vector vaccine as defined herein that expresses a protective antigen.

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of use, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Schematic depiction of the strategy for chromosomal integration of the antigen expression cassette P_(ompC)-gfpuv, encoding the model fluorescent antigen GFPuv. An osmotically-controlled GFPuv-encoding cassette (tandem white circle and hatched thick arrow) was constructed and linked to an aph marker encoding resistance to kanamycin (shaded thick arrow), flanked by FRT recombination sites (black triangles). The incoming P_(ompC)-gfpuv-aph cassette was integrated into the live vector chromosome using the λ Red recombination system, followed by removal of the aph marker using FLP recombinase, to yield the final live vector strain bearing no genes encoding resistance to antibiotics. The bacterial chromosome is represented by 5′-proximal and 3′-terminal darkened rectangles, and the black circle labeled with a “P” represents the wild-type chromosomally-encoded promoter of the deleted target open reading frame (e.g., guaBA or htrA).

FIG. 2. Flow cytometry histograms of GFPuv-mediated fluorescence encoded by P_(ompC)-gfpuv gene cassettes integrated into either the guaBA (thick solid line), htrA (thin hatched line), or clyA (thick broken line) sites of the attenuated S. Typhi live vector vaccine candidate CVD 910, compared to the vaccine strain alone (thin dotted line). Fluorescence intensities are measured for individual bacterial cells grown under inducing conditions of 200 mM NaCl in rich medium at 37° C./250 rpm for 16 hr.

FIG. 3. Schematic depiction of the strategy for chromosomal integration of the cell binding domain from toxin A of C. difficile. A synthetic codon-optimized gene cassette encoding the cell binding domain from toxin A designated 14cbd/a was prepared where the osmotically regulated P_(ompC) promoter was genetically fused to a promoterless 14cbd/a gene (tandem white circle and hatched thick arrow) and linked to an aph marker encoding resistance to kanamycin (shaded thick arrow), flanked by FRT recombination sites (black triangles). The incoming P_(ompC)-14cbd/a-aph cassette was integrated into the live vector chromosome using the λ Red recombination system, followed by removal of the aph marker using FLP recombinase, to yield the final live vector strain bearing no genes encoding resistance to antibiotics. The bacterial chromosome is represented by 5′-proximal and 3′-terminal darkened rectangles, and the black circle labeled with a “P” represents the wild-type chromosomally-encoded promoter of the deleted target open reading frame (e.g., guaBA, htrA or rpoS).

FIG. 4. Western immunoblot analysis. Six hour liquid broth cultures of CVD 910-2A (“2A”) were compared to cultures of CVD 910-3A (“3A”) under either inducing (200 mM NaCl to activate P_(ompC)) or non-inducing (15 mM NaCl) conditions.

FIG. 5. Schematical depiction of live vaccine strain CVD 910-3Assb(pSEC10-CBD/B).

FIG. 6. Comparison of hemolytic activity of fusion proteins expressed in CVD 910-4A, CVD 910-3A-GB2 and CVD 910-4Assb(pSEC10S2-B2). The noted strains were grown on trypticase soy agar with 5% sheep red blood cells under conditions of incubation at 37° C. for 24 hours. The plates were then photographed without magnification.

FIG. 7. Schematical depiction of live vaccine strain CVD 910-3A-GB2ssb (pSEC10-CBD/B) which contains insertions into the guaBA, htrA, rpoS and clyA loci and carries the plasmid pSEC10-CBD/B.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technical references.

As used herein, “a” or “an” may mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

As used herein, “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., +/−5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.

II. The Present Invention

Live vectors engineered for delivery of foreign antigens to the host immune system have performed well in experimental animal models, but have been only modestly successful in clinical trials. [20] Given the advances in the development of powerful plasmid-based expression technologies designed to deliver ample levels of foreign protein, it is unlikely that the lack of antigen-specific immunity observed in clinical trials is due to insufficient antigen synthesis following immunization. To the contrary, it is likely that inappropriate antigen synthesis occurring in vivo results in sufficient shock to the metabolism of the live vector to over-attenuate the strain and destroy immunogenicity. Although various attempts have been made to control the timing of foreign protein synthesis, using tightly regulated promoters to control transcription of genes in response to host environmental signals for example, improved immunogenicity in animals has not translated into improvements in clinical trials.[12,21,22]

A novel and elegant solution to this dilemma is presented here, wherein over-attenuation is circumvented by linking antigen synthesis to the growth rate of the live vector vaccine, such that synthesis is initially low after immunization, but steadily increases as the vaccine strain adjusts to prevailing environmental conditions and undergoes limited replication within the host. This expression strategy allows for efficient expression of one or even multiple foreign antigens within a single live vector vaccine strain. It can also be used in conjunction with plasmid-based methods by distributing the location of antigen expression cassettes between the chromosome and an expression plasmid. The approach presented herein thus offers the flexibility of independently adjusting the copy number of potentially toxic foreign genes by integrating a designated number of copies into the chromosome. By appropriate integration of foreign genes into chromosomal loci whose induction of expression is intimately associated with the physiology and growth rate of the vaccine strain, it becomes possible to “tune” foreign antigen synthesis to the metabolic state of the live vector.

The present invention is therefore based on the discovery that delivery of sufficiently immunogenic levels of chromosomally-encoded antigens to a subject can be accomplished through strategic integration of antigen expression cassettes into multiple locations within the live vector chromosome, thereby compensating for loss of copy number afforded by systems using stable low copy plasmids, while avoiding further attenuation of the vaccine strain. Integration of multiple cassettes also avoids the need for strong constitutive promoters to enhance antigen synthesis from a single gene copy, an approach which does not necessarily lead to adequate antigen synthesis or immune responses.[11,12]

The present invention is directed to several related embodiments, including (i) an attenuated strain of S. Typhi having disruptions of two or more chromosomal locations selected from the group consisting of the guaBA locus, the htrA locus, the clyA locus, the rpoS locus, and the ssb locus, and (ii) antigen-expressing attenuated strains of S. Typhi having a chromosomal-based expression system which comprises an antigen expression cassette integrated into two or more locations of chromosomal disruptions, (iii) antigen-expressing attenuated strains of S. Typhi having a chromosomal-based expression system which comprises an antigen expression cassette integrated into two or more locations of chromosomal disruptions as well as a plasmid-based expression system, (iv) a live vector vaccine comprising an antigen-expressing attenuated strain of S. Typhi as defined herein, and a pharmaceutically-acceptable carrier or diluent, (v) methods of inducing immune responses to an antigen of interest in a subject using the live vector vaccines as defined herein, and (vi) methods of vaccinating a subject using the live vector vaccines as defined herein.

Attenuated Strains of S. Typhi

As suggested above, in one embodiment the present invention is directed to attenuated strains of Salmonella enterica serovar Typhi having disruptions of two or more chromosomal locations. S. Typhi is a well-tolerated live vector that can deliver multiple unrelated immunogenic antigens to the human immune system. S. Typhi live vectors have been shown to elicit antibodies and a cellular immune response to an expressed antigen. S. Typhi is characterized by enteric routes of infection, a quality which permits oral vaccine delivery. S. Typhi also infects monocytes and macrophages and can therefore target antigens to professional APCs.

The genetic disruptions are sufficiently extensive to ensure that active forms of the protein(s) encoded by the locus harboring the disruption are not produced by the bacteria. While the skilled artisan will understand that the characteristics and scope of the disruptions can vary widely, in one non-limiting aspect the disruptions are sufficiently extensive to ensure that neither the protein(s) encoded by the locus, nor fragments thereof, can be detected in bacteria having the disruptions.

The skilled artisan will recognize that strains of bacteria having the disruptions can be readily produced via several techniques known in the art, including the λ Red-mediated site-directed mutagenesis method. [16] Other, less efficient, chromosomal deletion/integration technologies used in the past involve the use of suicide plasmids. These suicide plasmids exhibit replication which is exclusively dependent on the pir protein; successful deletions/integrations are dependent on recA-mediated homologous chromosomal crossovers, and counter-selection with sacB. [29]

The chromosomal locations having the two or more disruptions are the guaBA locus, the htrA locus, the clyA locus, the rpoS locus, and the ssb locus. The disruptions of these loci can be disruptions of the endogenous coding sequences, non-coding control sequence, promoter sequences, or a combination thereof. In the case of the guaBA locus, for example, the coding region can be disrupted without damaging the promoter sequence for the loci.

The chromosomal disruptions can include any combination of deletions or insertions of sequences comprising the disrupted loci.

The attenuated strains of S. Typhi may have disruptions in any combination of two, three or four of the loci and sites, or even all five of the loci.

Specific examples of such attenuated strains of S. Typhi including strain CVD 910, which contains disruptions in the guaBA locus and the htrA locus. Because the parent Ty2 strain used in the production of CVD 910 has the rpoS locus naturally inactivated, CVD 910 also contains a disruption of the rpoS locus. Thus, CVD 910 contains disruptions in three chromosomal locations: the guaBA locus, the htrA locus, and the rpoS locus.

Antigen-Encoding Attenuated Strains of S. Typhi

As suggested above, in a related embodiment the invention is directed to antigen-encoding attenuated strains of S. Typhi having a chromosomal-based expression system integrated into two or more of the disrupted chromosomal locations within the bacteria. Thus, the invention includes the attenuated strains of S. Typhi as defined herein, including strain CVD 910, which have been engineered to have antigen expression cassettes integrated into the locations of chromosomal disruptions.

The chromosomal-based expression systems used in the antigen-encoding attenuated strains of S. Typhi are simple in nature in that they comprises a genetic cassette (antigen expression cassette) comprising the coding sequence of an antigen of interest and, optionally, an exogenous promoter to direct transcription of the coding sequence. In some circumstances and depending on the identity of the coding sequence and/or promoter, additional 5′ and/or 3′ non-coding sequence associated with the coding sequence of the antigen of interest can be included in the cassette. Together, these sequences make up an antigen expression cassette that can be inserted into one or more disrupted bacterial chromosomes. Because the attenuated strains of S. Typhi defined herein have at least two chromosomal disruptions, antigen expression cassettes encoding different antigens can be used in the same strain of bacteria. In those strains of S. Typhi having three chromosomal disruptions, up to three different antigens may be expressed, with up to four different antigens in those strains of S. Typhi having four chromosomal disruptions, and up to five different antigens in those strains of S. Typhi having five chromosomal disruptions. The skilled artisan will also recognize that different combinations of antigens can be expressed in a given strain depending on the number of disruptions and the selected antigen expression cassettes. For example, where a strain has three disruptions, the same antigen expression cassette (encoding antigen A, for example) can be inserted into each of the three sites. Alternatively, an antigen expression cassette encoding antigen A could be inserted into two of the sites, while an antigen expression cassette encoding antigen B could be inserted into the third site. In a further alternative, an antigen expression cassette encoding antigen A could be inserted into one of the sites, an antigen expression cassette encoding antigen B could be inserted into the second site, and an antigen expression cassette encoding antigen C could be inserted into the third site. Thus, the identity of the antigen encoded by each antigen expression cassette is individually selected and any combination of antigens may be used. This expressly includes instances where a particular antigen is encoded by two or more cassettes as well as instances where a particular antigen is encoded by only one cassette.

Antigens

The antigen expression cassettes of the present invention preferably express an antigen for presentation to a host to elicit an immune response resulting in immunization and protection from disease. The antigens of interest that may be expressed in the attenuated strains of S. Typhi of the present invention are unlimited in identity and include, but are not limited to, antigens from foreign bacteria (e.g., proteins not already expressed by S. Typhi), viruses, and parasitic organisms. Exemplary antigens are protective antigens (i.e., an antigen that when bound by an antibody, result in the death of the organism expressing the antigen). Because the attenuated strains of S. Typhi may be used as live vector vaccines, one practicing the invention will be motivated, in one aspect, to use antigens suspected of or known to induce a protective immune response in a subject. As another example, antigens suspected of or known to induce an active immune response in a subject to pre-existing infection may also be used.

Exemplary antigens that can be used to induce a protective immune response include detoxified versions of enterotoxin A (TcdA), enterotoxin B (TcdB), and binary toxin (transferase; Cdt) of Clostridium difficile, and fragments thereof, such as the cell-binding domains of TcdA and TcdB, and the binding domain of Cdt (CdtB). These antigens can be used to produce a mono-, bi- or multivalent live vector vaccine that in turn can be used to vaccinate a subject against infections caused by C. difficile. With the ability to impart immunity that recognizes both toxins and putative colonization factors, a subject can be vaccinated against disease that occurs at two critical stages of infection—colonization and toxin production.

Additional exemplary antigens include the LcrV antigen and the capsular F1 antigen of Yersinia pestis, which are required for secretion of virulence effectors proteins and are virulence factors themselves. Additional Yersinia pestis antigens include pH 6 antigen (Psa), a putative colonization factor, and Yop B/YopD, two essential proteins comprising the translocon region of the type 3 secretion (T3SS) needle.

Given the ease with which antigen expression cassettes can be produced, and the straightforwardness of inserting and removing the cassettes from locations of chromosomal disruptions in bacteria, it will be clear to the skilled artisan that a very wide range of different antigens can be expressed using the chromosomal-based expression systems of the attenuated strains of S. Typhi defined herein. These same antigens can also be expressed in the bacteria using the plasmid-based expression systems discussed below.

Plasmid-Based Expression Systems

In a related embodiment, the invention is also directed to antigen-encoding attenuated strains of S. Typhi having the chromosomal-based expression system discussed above, as well as a plasmid-based expression system. The inclusion of a plasmid-based expression system within the bacteria provides additional flexibility for expressing antigens of interest in the attenuated strains of S. Typhi of the invention. The plasmid-based expression system allows expression of additional copies of an antigen expression cassette that is integrated into the bacterial chromosome or an antigen expression cassette encoding an antigen of different identity. Thus, as defined herein the antigen expression cassettes can be inserted to both the locations of chromosomal disruptions (in terms of the chromosomal-based expression systems) and expression plasmids (in terms of the plasmid-based expression systems).

SSB-Stabilized Plasmid-Based Expression System

Examples include the plasmid-based expression systems disclosed in U.S. Pat. Nos. 6,703,233, 6,969,513, 6,977,176, 7,141,408, 7,125,720 and 7,138,112, each of which is incorporated herein by reference in their entirety. The systems described in these patents are multicopy expression plasmids into which plasmid maintenance systems have been incorporated. Such multicopy expression plasmids produce a gene dosage effect which enhances the level of expression of the antigen of interest. In a specific example, a plasmid-based expression system has been developed in which a low copy expression plasmid has been engineered to encode an essential single-stranded binding protein (SSB) which has been deleted from the bacterial vaccine strain. Since SSB is essential for DNA replication, recombination, and repair, ssb-deleted bacteria must maintain the expression plasmid to enable survival. Therefore, if SSB-stabilized plasmids are used to encode one or more foreign antigens, then bacteria become committed to foreign antigen synthesis. If expression of foreign antigens in the cytoplasm of the bacteria becomes toxic, antigen export systems can be further introduced into these SSB-stabilized plasmids to export fusion proteins out of the cytoplasm and minimize metabolic disruption.

ClyA Fusion Protein Plasmid-Based Expression Systems

A further example of a suitable plasmid-based expression system is disclosed in WO 09/149083, incorporated herein by reference in its entirety, which makes use of the S. Typhi HlyE family of export proteins, including the cryptic hemolysin (ClyA), encoded by the cytolysin A gene (clyA). ClyA from S. Typhi was first described by Wallace et al. who also reported the crystal structure for the homologous hemolysin from E. coli. [26] This hemolysin has been described previously and variously referred to as ClyA, HlyE, or SheA.

The crystal structure of ClyA in E. coli has been resolved. [26] The unique structure can be roughly divided into several domains, a head domain, a body domain and a tail domain. The body domain consists of a bundle of helixes (A, B, C, D, F). The tail domain is a helix G which extends to half the length of the body. The head domain consists of a short β hairpin (β-tongue) and two small helices (D and E), each flanking the β-tongue. It was suggested that the β-tongue might be critical for pore formation and hence for the hemolytic activity. [26] Through site directed mutagenesis, it was found that many regions of ClyA were important for the hemolytic activity. [27]

The ClyA protein is exported from both E. coli and S. Typhi and it is capable of exporting passenger proteins (and antigens of interest) that have been genetically fused to the 3′-terminus of the clyA open reading frame. It is demonstrated that the proper folding of these fusion proteins occurs such that the inherent biological activity of the domains involved is maintained.

The amino acid and nucleotide sequence for the isolated S. Typhi clyA gene and ClyA protein (from Salmonella serovar Typhi strain Ty2) are provided as SEQ ID NO:39 and SEQ ID NO:38, respectively. A synthetic codon-optimized version of the S. Typhi clyA gene, as described and utilized herein, is provided in SEQ ID NO:40. Other HlyE family members that may be utilized as export proteins herein are also available and known to those of ordinary skill in the art. The family members include a second S. Typhi cytolysin A (the clyA gene is set forth in SEQ ID NO:41 and it is available under GENBANK Accession No. AJ313034); Salmonella paratyphi cytolysin A (the clyA gene sequence for cytolysin A is set forth in SEQ ID NO:42 and it is available under GENBANK Accession No. AJ313033); Shigella flexneri truncated HlyE (the hlyE gene sequence is set forth in SEQ ID NO:43 and it is available under GENBANK Accession No. AF200955); Escherichia coli HlyE (the hlyE gene sequence is set forth in SEQ ID NO:44 and it is available under GENBANK Accession No. AJ001829).

As indicated above, the HlyE family of proteins typically causes cytolysis of target cells, including hemolysis of erythrocytes. Because cytolysins/hemolysins may be considered to be virulence factors, the present invention encompasses the use of variants of HlyE family members that have been mutated such that they lack, or have reduced, hemolytic activity. The ability of these variants to be exported from a bacterial cell producing them, alone or in the context of fusion to a protein of interest, has been maintained. Thus, the non-hemolytic variants of HlyE family members have reduced or no hemolytic activity, and yet are fully functional in the plasmid-based expression systems of the present invention. Such variants include the S. Typhi cytolysin A (ClyA) protein of SEQ ID NO:38 having a single mutation selected from the group consisting of an 5195N mutation, an I198N mutation, an A199D mutation, an E204K mutation, and a C285W mutation; an I198N, C285W double mutation; and an I198N, A199D, E204K triple mutation. The S. Typhi cytolysin A (ClyA) protein may also have the amino acid sequence set forth in SEQ ID NO:38 and a C285W mutation, as well as one additional mutation selected from the group consisting of an I198N mutation, an A199D mutation, and an E204K mutation.

The plasmid-based expression systems comprising ClyA fusion proteins described herein can be used to express and export a wide variety of fusion proteins comprising an export protein and an antigen of interest. The export protein::antigen of interest fusion protein construct is present in an antigen expression cassette, which in turn is present in an expression plasmid to facilitate the recombinant production of the protein of interest. Typically the expression plasmid will comprise an origin of replication and other structural features that control and regulate the maintenance of the expression plasmid in the host cell. Exemplary expression plasmids are well known to the skilled artisan.[7,23,28] The key aspect of such expression plasmids is copy number, which can range from several hundred per chromosomal equivalent to one per chromosomal equivalent. Preferably the copy number of the expression plasmids is between 5 and 15 copies per chromosomal equivalent.

Live Vector Vaccines

As suggested above, and in a related embodiment, the invention is directed to a live vector vaccine comprising one or more of the antigen-encoding attenuated strains of S. Typhi as defined herein, and a pharmaceutically-acceptable carrier or diluent.

It is contemplated that the live vector vaccines of the present invention will be administered as pharmaceutical formulations for use in vaccination of individuals, preferably humans. In addition to the strains of S. Typhi, the vaccines will thus include pharmaceutically-acceptable carriers, and optionally, may include other therapeutic ingredients, such as various adjuvants known in the art.

The carrier or carriers must be pharmaceutically acceptable in the sense that they are compatible with the therapeutic ingredients and are not unduly deleterious to the recipient thereof. The therapeutic ingredient or ingredients are provided in an amount and frequency necessary to achieve the desired immunological effect.

The mode of administration and dosage forms will affect the therapeutic amounts of the compounds which are desirable and efficacious for the vaccination application. However, the live vector vaccines are delivered in an amount capable of eliciting an immune reaction in which it is effective to increase the subject's immune response to the antigen(s) of interest. An immunogenic amount is an amount which confers an increased ability to prevent, delay or reduce the severity of the onset of a disease, as compared to such abilities in the absence of such immunization. It will be readily apparent to one of skill in the art that this amount will vary based on factors such as the weight and health of the recipient, the type of antigen(s) being expressed, the type of infecting organism being combatted, and the mode of administration of the vaccines.

The vaccines may be formulated for any suitable means and/or methods for delivering the live vector vaccines to a corporeal locus of the subject where the live vector vaccines are intended to be effective in triggering an immune response, for example, for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, pulmonary, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), and intramuscular (i.m.). Any known device useful for parenteral injection or infusion of vaccine formulations can be used to effect such administration. In preferred aspects of each of the embodiments on the invention, the vaccines are administered to a subject as an oral formulation, in particular, to the oral mucosa.

The dose rate and suitable dosage forms for the live vector vaccines of the present invention may be readily determined by those of ordinary skill in the art without undue experimentation, by use of conventional antibody titer determination techniques and conventional bioefficacy/biocompatibility protocols. Among other things, the dose rate and suitable dosage forms depend on the particular antigen employed, the desired therapeutic effect, and the desired time span of bioactivity.

Formulations of the vaccines can be presented, for example, as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the vaccine; or as a suspension.

Depending on the means of administration, the vaccines may be administered all at once, such as with an oral formulation in a capsule or liquid, or slowly over a period of time, such as with an intramuscular or intravenous administration. The vaccines may also be administered to the subject more than once, as boosters, for example, where administration of separate doses of the vaccines may be separated in time by hours, days, weeks or months.

In each embodiment and aspect of the invention, the subject is a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.

Manners of Use

As indicated above, it is intended that the antigen-encoding attenuated strains of S. Typhi defined herein will be grown under conditions that may induce expression of the antigens of interest prior to immunization, and formulated as a live vector vaccine for administration to a subject, whereupon an immune response to the antigens of interest, inter alia, will be induced in the subject.

The invention therefore includes methods of inducing an immune response to an antigen of interest in a subject, comprising administering to a subject a live vector vaccine as defined herein and that expresses an antigen of interest. The invention also includes methods of vaccinating a subject with a protective antigen, comprising administering to a subject a live vector vaccine as defined herein that expresses a protective antigen.

The methods contemplate and include administering the live vector vaccine to the subject only once, or more than once, such as 2, 3, 4, 5 or more times.

A non-limiting example of the manner in which the vaccines may be used includes use of the vaccine as a nosocomial oral vaccine, administered to patients seven days after antibiotic treatment for Clostridium difficile infection (CDI) to block recurrent disease by eliciting a vigorous and rapid anamnestic response in patients primed by the initial C. difficile infection.

IV. Examples Materials and Methods

Bacterial Strains and Culture Conditions.

The attenuated S. enterica serovar Typhi (S. Typhi) live vector vaccine strain CVD 910 used in these studies is an auxotrophic derivative of wild-type strain Ty2, with deletions in guaBA and htrA. To improve the clinical acceptability of the live vector vaccine strains, all genetic and bacteriologic manipulations of the live vectors were performed using an animal product-free medium equivalent to Luria-Bertani medium, comprised of 10 g/liter of Soytone (Teknova; S9052), 5 g/liter Hy-Yest 412 (Sigma; Y1001), and 3 g/liter NaCl (American Bioanalytical; AB01915), supplemented with 0.002% guanine (Sigma; G6779).

Construction of Chromosomal Integrations.

Deletion cassettes were constructed for use with the λ Red-mediated site-directed mutagenesis method [16] to delete either guaBA, htrA, or clyA from wild-type S. Typhi Ty2. Cassettes encoding upstream and downstream flanking chromosomal sequences were constructed using primer pairs listed in Table 1 and purified chromosomal DNA from Ty2 as the template DNA.

TABLE 1 Primers used in the construction and testing of live vector strains expressing chromosomally-encoded GFPuv. Primer (SEQ ID NO:) Sequence^(a) 5guaBA-for 5′- GAATTCTAGCTGCTCATACTTCTGCTGCA -3′ SEQ ID NO: 1 5guaBA-rev 5′- GCTAGCCAATTGGGGCAATATCTCACCTGG -3′ SEQ ID NO: 2 3guaBA-for 5′- GGATCCACTAGTGTCGATAACCCTTCCTGTGT -3′ SEQ ID NO: 3 3guaBA-rev 5′- CTCGAGACAGCACCTACAAGTCTGGCATG -3′ SEQ ID NO: 4 guaBA PCR-for 5′- GCGCTGACCACCGGAATACGGCTG -3′ SEQ ID NO: 5 guaBA PCR-rev 5′- CATGGCATGGATGAGGCAACCGCGAAGC -3′ SEQ ID NO: 6 5htrA-for 5′- GAATTCGTACCTTCAATCAGGCGTTACTGGAAGATG -3′ SEQ ID NO: 7 5htrA-rev 5′- GCTAGCCAATTGCGATTAACAGGTAACGCAAAATTGCTGTGTACGTCAG -3′ SEQ ID NO: 8 3htrA-for 5′- GGATCCACTAGTCTGCGTAAGATTCTCGACAGCAAGCCGTCGGT -3′ SEQ ID NO: 9 3htrA-rev 5′- CTCGAGCCAGCATCATTTCGGCAGTCATACACACCAGTTCGC -3′ SEQ ID NO: 10 htrA PCR-for 5′- GTGTCGCCGATCTTGAAGACGCGGTAGAG -3′ SEQ ID NO: 11 htrA PCR-rev 5′- CTATCGACGCCAAGCTGGCCGCTGTCGAC -3′ SEQ ID NO: 12 5clyA-for 5′- TAGTAATGAGAATTCGCTGGTATTGATCGGCTCTCCGGTAGAGATTAGCGA -3′ SEQ ID NO: 13 5clyA-rev 5′- GCTAGCCAATTGTGCCTCTTTAAATATATAAATTGCAATTAAGTACCTG -3′ SEQ ID NO: 14 3clyA-for 5′- GGATCCACTAGTGATACATTTTCATTCGATCTGTGTACTTTTAACGCCCGAT SEQ ID NO: 15 AGCG -3′ 3clyA-rev 5′- TGATAGTAACTCGAGACAATCCATAAGAAAGGTCAGGCACACTGGGAAGG SEQ ID NO: 16 CGACATC -3′ clyA PCR-for 5′- CATGATGGTATCCAGTATGGCACAAGC -3′ SEQ ID NO: 17 clyA PCR-rev 5′- GTAATCGACAACATGCTACATCCATCG -3′ SEQ ID NO: 18 5FRT-aph-for 5′- GAATTCGCTAGCGCTGGAGCTGCTTCGAAGTTC -3′ SEQ ID NO: 19 3FRT-aph-rev 5′- CTCGAGTTCCGGGGATCCGTCGACCTGCAGTTC -3′ SEQ ID NO: 20 5gfpuv 5′- CAATTGTGTGGTAGCACAGAATAATGAAAAGT -3′ SEQ ID NO: 21 3gfpuv 5′- GCTAGCTCATTATTTGTAGAGCTCATCCAT -3′ SEQ ID NO: 22 ^(a)Relevant restriction sites are underlined.

These cassettes were used to exchange chromosomal targets with a Tn5 neomycin phosphotransferase cassette (aph), encoding resistance to kanamycin, and recombined into the chromosome using the λ Red recombination system encoded by pKD46. Final removal of the kanamycin resistance cassette was accomplished using FLP recombinase encoded by pCP20. The integrity of the intended chromosomal deletion mutations was confirmed by DNA sequence analysis of the chromosomal locus from each strain using PCR primers listed in Table 3. For chromosomal expression of GFPuv, an antigen expression cassette in which an osmotically regulated ompC promoter (P_(ompC) [23]) was linked to gfpuv was selected and inserted 5′-proximal to the aph resistance marker of chromosomal deletion cassettes. As shown in FIG. 1, care was taken to preserve the natural chromosomal promoters controlling transcription of chromosomally encoded targets, with the intent that synthesis of GFPuv would ultimately be controlled both by osmolarity (via P_(ompC)) as well as growth rate in the case of the guaBA locus,[15] heat shock/environmental stress in the case of the htrA locus,[18] or possibly low pH for clyA. [19]

Flow Cytometry.

GFPuv-expressing strains were grown overnight at 37° C. on rich solid medium supplemented with guanine. 2-3 fluorescing colonies were then inoculated into 20 ml of supplemented liquid medium and incubated with shaking at 250 rpm overnight at 37° C. Overnight starter cultures were then diluted 1:100 into fresh supplemented liquid medium and incubated at 37° C., 250 rpm. For growth curve studies, 5 ml volumes were periodically removed from incubating cultures, from which bacteria from 4 ml were pelleted, while the remaining 1 ml volume was used to measure the optical density at 600 nm (OD₆₀₀). Pelleted bacteria were resuspended in 1 ml of PBS, and cells then diluted 1:1,000 in PBS prior flow analysis. Quantitation of GFPuv fluorescence was analyzed using a MoFlo Legacy flow cytometer/cell sorter system (Beckman Coulter) with the argon laser exciting bacteria at 488 nm and emissions detected at 525 nm. Forward versus side light scatter, measured with logarithmic amplifiers, was used to gate on bacteria. A minimum of 50,000 events were acquired from each sample at a collection rate of approximately 3,500 events per second. The mean fluorescence intensity was determined using Summit software (Beckman Coulter). Background autofluorescence was determined using the negative control S. Typhi vaccine strain CVD 910.

Results

Construction of CVD 910.

The attenuated vaccine candidate CVD 908-htrA, derived from Ty2 and carrying deletions in aroC, aroD, and htrA, was previously constructed and proved to be safe and highly immunogenic in Phase 2 clinical trials.[13] Here, a new vaccine strain, CVD 910, was constructed that carries deletions in guaBA and htrA. The ΔaroC ΔaroD was replaced with the single deletion ΔguaBA for two important reasons: 1) previous work by the inventors showed that ΔguaBA alone sufficiently attenuates Ty2, resulting in a live vector strain capable of eliciting impressive humoral immunity to a plasmid-encoded foreign antigen using the murine intranasal model of immunogenicity;[14] and 2) transcriptional control of the guaBA locus is controlled by growth rate, independent of guanine-mediated repression,[15] allowing expression of properly integrated antigen expression cassettes to be increased as the live vectors grow in the host. In order to reduce the risk of reversion to virulence by the unlikely acquisition of wild type guaBA genes, a secondary deletion of htrA which encodes a heat shock-induced serine protease was further engineered.

Deletion cassettes targeting guaBA and htrA were constructed for use with the λ Red-mediated site-directed mutagenesis method,[16] and each cassette was used to successfully delete either guaBA or htrA from wildtype S. Typhi Ty2. Introduction of both deletion mutations into a single strain resulted in the creation of CVD 910. A preliminary assessment of attenuation of CVD 910 was carried out by comparing the minimum lethal dose causing death in 50% of a group of BALB/c mice (LD50) for CVD 910 versus CVD 908-htrA, using the hog gastric mucin intraperitoneal murine challenge model. For this model, the guidelines recommended in the Code of Federal Regulations for Food and Drugs, Title 21, Part 620.13 (c-d), 1986 for intraperitoneal challenge of mice with S. Typhi were broadly followed. Using this method, the LD50 for both CVD 910 and CVD 908-htrA was determined to be approximately 5×10⁵ CFU (data not shown), versus an LD50 of ˜10 CFU for wild-type Ty2,[17] demonstrating construction of a novel live vaccine strain with a safety profile equivalent to that of CVD 908-htrA.

Chromosomal Integration of Gfpuv Cassettes into CVD 910.

GFPuv was expressed from independently controlled cassettes in CVD 910 (containing the guaBA and htrA chromosomal gene deletions) in the following manner. The osmotically regulated P_(ompC) promoter was genetically fused to a promoterless gfpuv gene. The resulting P_(ompC)-gfpuv cassette was integrated into either the guaBA or htrA loci such that only the open reading frame was replaced, but the original promoters for both chromosomal loci were preserved, as depicted schematically in FIG. 1. For example, integration of P_(ompC)-gfpuv into the guaBA locus to create CVD 910-GG resulted in transcription of gfpuv controlled both by osmolarity (via P_(ompC)) and growth rate (via P_(guaBA)). Similarly, integration of the same cassette into htrA to create CVD 910-HG resulted in synthesis of GFPuv controlled both by osmolarity (P_(ompC)) and heat shock/environmental stress (P_(htrA)).[18] In addition, a third chromosomal integration was prepared, CVD 910-CG, in which P_(ompC)-gfpuv replaced clyA, encoding a cryptic hemolysin from Ty2 whose transcription is normally controlled by low pH.[19] Interestingly, when the resulting strains were grown overnight at 37° C. in liquid cultures and analyzed for fluorescence by flow cytometry, observed fluorescence intensity was found to be strongly influenced by the site of integration, regardless of osmotic induction of P_(ompC). As shown in the fluorescence histograms of FIG. 2, under inducing conditions of 200 mM NaCl, strains with P_(ompC)-gfpuv integrated into either guaBA or htrA displayed remarkably uniform bacterial populations with mean fluorescence intensities of 28.65 and 21.59 respectively, while integration into clyA resulted in a very low mean fluorescence intensity of 7.53, barely above the background autofluorescence of 5.94 detected for CVD 910 alone. Having established substantial expression of GFPuv from two independent chromosomal loci, the hypothesis that integration of P_(ompC)-gfpuv into both guaBA and htrA together would result in additive expression of fluorescence was then tested. Analysis of fluorescence from the resulting strain, CVD 910-2G, revealed an uninduced (50 mM NaCl) mean fluorescence intensity of 36.01, which increased to 48.21 after induction with 200 mM NaCl. In this experiment, uninduced fluorescence intensities for CVD 910-GG and CVD 910-HG were 25.35 and 15.85 respectively, while induced fluorescence levels were 32.46 and 24.03 respectively. It is immediately evident that for overnight liquid cultures, cumulative fluorescence observed with two copies of gfpuv integrated into CVD 910-2G is approximately equivalent to the combined fluorescence levels for individual copies of integrated gfpuv observed in CVD 910-GG and CVD 910-HG, under both uninduced and induced osmotic conditions.

Growth-Phase Regulated Expression of GFPuv in CVD 910-2G.

Regulated, but sustained, expression of foreign antigens delivered by live vectors is expected to reduce any metabolic burden associated with antigen synthesis, thereby allowing live vectors to persist longer in immunized hosts and prolong delivery of candidate vaccine antigens to the immune system. [20] However, and despite recent improvements, tightly regulated and appropriately timed antigen expression using plasmid-based expression technologies still remains elusive in many cases, with leaky expression potentially contributing to over-attenuation of live vector vaccine strains. Therefore, one of the goals of the current work was to investigate the feasibility of linking foreign antigen expression to the growth phase of the live vector, such that expression would be reduced when bacteria are adapting to a significant change in environmental conditions (i.e. lag phase), but would be strongly induced after bacteria have successfully adapted their metabolism to new energy sources and environmental conditions (i.e. exponential growth transitioning into stationary phase).

To meet this goal, chromosomally-encoded GFPuv expression in CVD 910-2G was first compared to a previously described live vector CVD 908-htrAssb(pGEN206) [3], in which GFPuv was expressed independently of growth phase from a low copy (˜5 copies per chromosomal equivalent) stabilized expression plasmid. Overnight starter cultures of CVD 910-2G and CVD 908-htrAssb(pGEN206) were grown at 37° C. for approximately 16 hrs and then diluted 1:100 into 100 ml of fresh medium in 250 ml baffle flasks. To reduce the influence of osmolarity on growth phase and more clearly establish any link between observed fluorescence and induction of P_(guaBA) and P_(htrA) during growth, all strains were grown under non-inducing conditions of 50 mM NaCl. Fresh cultures were incubated at 37° C./250 rpm, and 5 ml aliquots were removed every hour for 6 hours to measure both OD₆₀₀ and fluorescence intensities by flow cytometry. As expected, plasmid-based expression in CVD 908-htrAssb(pGEN206) significantly slowed the growth kinetics of the live vector when compared to either CVD 910 or CVD 910-2G, even under non-inducing conditions of 50 mM NaCl (Table 2). Initial fluorescence intensities in lag phase started out quite high at 1262.66, dipped during exponential phase to 686.27, and then rose again to 1131.59 in stationary phase. In sharp contrast, the kinetics of GFPuv expression in CVD 910-2G was closely linked to the growth phase of the culture, with a low mean fluorescence intensity of 81.19 measured in the lag phase, which gradually increased with cell density to a maximum fluorescence intensity of 200.06 as the culture reached stationary phase. The observed variation of fluorescence with growth phase, as quantitated by flow cytometry, is not an aggregate effect of increasing cell numbers, but instead reflects the level of GFPuv synthesis within individual bacteria in a growth-rate dependent manner. These data support the feasibility of chromosomal expression of a foreign antigen from multiple integration sites, and the possibility of antigen expression synchronized with growth-rate, a possibility not supported by plasmid-based expression in these experiments.

TABLE 2 Chromosomal versus plasmid-based expression of GFPuv in attenuated Salmonella Typhi live vectors. CVD 910-2G CVD 908htrAssb Time CVD 910 (guaBA::gfpuv htrA::gfpuv) (pGEN206S2) (hr) OD₆₀₀ ^(a) MFI^(b) OD₆₀₀ MFI OD₆₀₀ MFI 0 0.04  ND^(c) 0.04 ND 0.04 ND 1 0.07 ND 0.08 81.19 0.06 1262.66 2 0.27 ND 0.3 96.77 0.14 1196.59 3 0.71 ND 0.71 105.59 0.38 721.34 4 1.36 ND 1.36 182.77 0.72 686.27 5 1.88 ND 1.86 ND 1.25 ND 6 2.18 6.34 2.18 169.87 1.67 891.53 7 2.29 ND 2.29 200.06 1.95 1131.59 ^(a)Cultures grown under non-inducing conditions in 50 mM NaCl. ^(b)Mean Fluorescence Intensity. ^(c)Not Determined.

This experiment was repeated to compare GFPuv expression from double integrations in CVD 910-2G to single integration expression levels in CVD 910-GG and CVD 910-HG. As summarized in Table 3, growth phase-dependent expression of fluorescence intensity was again observed, increasing from an initial lag phase level of 32.90 to a high of 161.65 in stationary phase. Interestingly, fluorescence levels during the 3 hr lag phase for the double integration did not reflect the sum of fluorescence observed with single integrations during this period, but became additive as the cultures progressed into exponential and stationary phases. Fluorescence intensities from single integrations did not seem to reflect the same dependence on growth phase as observed for the double integration; intensities for the guaBA integration in CVD 910-GG progressed from 74.94 to 96.31 during growth while htrA-controlled fluorescence in CVD 910-HG progressed from 32.90 to 68.94. Despite this anomaly, the data reported here suggest that integration of antigen expression cassettes into multiple loci within a live vector chromosome can be accomplished without further attenuation of the vaccine strain, and that this multiple integration strategy results in superior expression levels of foreign antigens versus conventional integration into a single locus.

TABLE 3 Growth-phase regulated chromosomal expression of GFPuv in CVD 910 attenuated Salmonella Typhi live vectors. CVD 910-2G CVD 910-GG CVD 910-HG (guaBA::gfpuv Time CVD 910 (guaBA::gfpuv) (htrA::gfpuv) htrA::gfpuv) (hr) OD₆₀₀ ^(a) MFI^(b) OD₆₀₀ MFI OD₆₀₀ MFI OD₆₀₀ MFI 0 0.04  ND^(c) 0.04 ND 0.03 ND 0.02 ND 1 0.09 ND 0.09 74.94 0.06 32.9 0.06 38.31 2 0.33 ND 0.3 71.03 0.24 49.08 0.24 72.53 3 0.81 ND 0.72 70.58 0.68 56.12 0.6 95.41 4 1.45 ND 1.31 75.26 1.29 60 1.36 121.95 5 1.96 ND 1.86 84.81 1.84 66.55 1.86 138.01 6 2.24 5.87 2.17 96.31 2.19 68.94 2.16 161.65 ^(a)Cultures grown under non-inducing conditions in 50 mM NaCl. ^(b)Mean Fluorescence Intensity. ^(c)Not Determined.

Construction and Testing of CVD 910-3A.

An additional strain of CVD 910 was prepared that expresses the cell binding domains from toxin A (CBD/A) or from toxin B (CBD/B) of C. difficile. A synthetic codon-optimized gene cassette encoding the cell binding domain from toxin A designated 14cbd/a was prepared where the osmotically regulated P_(ompC) promoter was genetically fused to a promoterless 14cbd/a gene. All P_(ompC)-controlled antigen cassettes encoding C. difficile antigens were constructed by inserting synthetic codon-optimized genes (encoding the cell binding domains of either 14CBD/A (SEQ ID NO:23) or CBD/B (SEQ ID NO:24)) as NheI-AvrII fragments into pSEC10 digested either with SpeI-NheI to generate the unfused P_(ompC)-14cbd/a encoding 14CBD/A, or pSEC10 cleaved only with NheI to generate the fused P_(ompC)-clyA::cbd/b encoding ClyA-CBD/B. In the case of P_(ompC)-14cbd/a, the resulting cassette was then excised from pSEC10 as an EcoRI-AvrII fragment and inserted into chromosomal integration cassettes in preparation for crossing into the chromosome using previously published λ Red integration technologies (see FIG. 3) [3,16]. All integration cassettes were integrated such that only the open reading frame of either guaBA or htrA was replaced, but the original promoters for both chromosomal loci were preserved, as depicted schematically in the chromosomal integration strategy of FIG. 3. For example, integration of P_(ompC)-14cbd/a into the guaBA locus resulted in transcription of 14CBD/A controlled both by osmolarity (via P_(ompC)) and growth rate (via P_(guaBA)). Similarly, integration of the same cassette into htrA resulted in synthesis of 14CBD/A antigen controlled both by osmolarity (P_(ompC)) and heat shock/environmental stress (P_(hfrA)).[18]

In addition, advantage was taken of the fact that all strains derived from Ty2 are naturally inactivated at the rpoS locus [24] in order to integrate a third copy of P_(ompC)-14cbd/a into the chromosome of CVD 910 without further attenuation of the live vector vaccine. Integration of P_(ompC)-14cbd/a into rpoS resulted in expression of 14CBD/A antigen controlled by osmolarity (P_(ompC)) and entry of growing vaccine organisms into stationary phase (P_(rpoS)) [25]. Additional primers used to construct the rpoS-targeted integration cassettes are listed below in Table 4.

TABLE 4 Primers used in the construction and testing of live vector strains expressing chromosomally encoded 14CBD/A from the rpoS locus. Primer (SEQ ID NO:) Sequence^(a) 5rpoS-for 5′- AAGCTTGAATTCCGTATTCTGAGGGCTCAGGTGAACAAAGTGC -3′ SEQ ID NO: 25 5rpoS-rev 5′- CCTAGGCAATTGACCCGTGATCCCTTGACGGAACTAGCAAGTC -3′ SEQ ID NO: 26 3rpoS-for 5′- GGATCCGGTTCGGTATCGCGCCAGGTATACAGACAATGC -3′ SEQ ID NO: 27 3rpoS-rev 5′- CTCGAGCCGGAAGTGCAGGCGGTAAACGCTATGTACAC -3′ SEQ ID NO: 28 rpoS PCR-for 5′- ATGCAGCACAGCAAGGAGTTGTGACCA -3′ SEQ ID NO: 29 rpoS PCR-rev 5′- GGTGCGTATCGATAAGGTCTCTTACCACAGC -3′ SEQ ID NO: 30 ^(a)Relevant restriction sites are underlined.

Successful integration of P_(ompC)-14cbd/a into guaBA, htrA, and rpoS, creating the live vector strain CVD 910-3A, was verified by direct chromosomal sequencing and listed here as SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; the protein amino acid sequence for 14CBD/A is listed as SEQ ID NO:34. In all chromosomal sequences presented, the location of the P_(ompC) promoter region, sequences encoding 14CBD/A, and residual FRT chromosomal scar sequences (left behind after removal of the kanamycin resistance marker) shown in Table 5. The location of key restriction sites (BamHI: GGATCC and XbaI: TCTAGA) are also shown in the Table as points of reference to be related back to the chromosomal integration strategy shown in FIG. 3.

TABLE 5 P_(ompC) Antigen residual FRT BamHI: XbaI: NheI: promoter coding chromosomal GGATCC TCTAGA GCTAGC region region scar sequences site site site SEQ ID 10-984 991-996 13-18 NO: 23 14CBD/A SEQ ID 10-1617 1624-1629 13-18 NO: 24 CBD/B SEQ ID 876-1361 1388-2362 2437-2470  1362-1367; 2152-2157 NO: 31 14CBD/A 2486-2491 SEQ ID 830-1315 1342-2316 2391-2424  1316-1321; 2406-2411 NO: 32 14CBD/A 2440-2445 SEQ ID 655-1140 1167-2141 2231-2264  1141-1146; 2246-2251 NO: 33 14CBD/A 2280-2285 SEQ ID 1-325 NO: 34 14CBD/A SEQ ID 1431-3029 699-732 714-719 NO: 35 14CBD/A SEQ ID  1-489 1431-3029  516-1430  490-495; 1425-1430 NO: 36 14CBD/A 3084-3089 SEQ ID 306-838  1-305 NO: 37 14CBD/A

Copy number-dependent osmotically controlled expression of 14CBD/A was confirmed by western immunoblot analysis. As shown in FIG. 4, six hour liquid broth cultures of CVD 910-2A (carrying P_(ompC)-14cbd/a integrated into guaBA and htrA) were compared to cultures of CVD 910-3A (carrying P_(ompC)-14cbd/a integrated into guaBA, htrA, and rpoS). All cultures were grown at 37° C. under either inducing (200 mM NaCl to activate P_(ompC)) or non-inducing (15 mM NaCl) conditions. Induction of 14CBD/A synthesis is clearly observed, with maximum expression confirmed for CVD 910-3A induced with high osmolarity.

Construction of CVD 910-3Assb(pSEC10-CBD/B).

A chromosomal deletion of ssb was introduced into CVD 910-3A as previously described [3], accompanied by introduction of the non-antibiotic SSB-stabilized expression plasmid pSEC10 into which a synthetic codon-optimized gene cassette encoding the cell binding domain of C. difficile toxin B was inserted. The resulting live vaccine strain, designated CVD 910-3Assb(pSEC10-CBD/B) is depicted schematically in FIG. 5. Confirmation of the chromosomal deletion of ssb as intended was confirmed by direct chromosomal sequencing as listed in SEQ ID NO:35; the integrity of the plasmid-based P_(ompC)-clyA-cbd/b cassette was also confirmed by direct sequencing as listed in SEQ ID NO:36, with the predicted amino acid sequence of ClyA-CBD/B listed in SEQ ID NO:37. Here again, for SEQ ID NO:35, the location of the residual FRT chromosomal scar sequences (replacing the deleted ssb gene) is shown in Table 5 along with the location of the internal XbaI site (TCTAGA). For the SEQ ID NO:36 sequence encoding ClyA-CBD/B, the location of the P_(ompC) promoter region is also shown in Table 5 along with the locations of the sequence encoding CBD/B and the key restriction sites (BamHI: GGATCC, NheI: GCTAGC, and AvrII: CCTAGG).

Proof-of-Principle Immunogenicity and Challenge Experiment Using a CVD 910 Bivalent Plague Vaccine.

The strategy for development of CVD 910-3Assb(pSEC10-CBD/B) was informed by a critical proof-of-principle experiment in which a bivalent live vector vaccine against pulmonary plague caused by Yersinia pestis was constructed and tested. Using the identical genetic engineering strategy used to create CVD 910-3Assb(pSEC10-CBD/B), a bivalent CVD 910-based plague vaccine was constructed that expressed the full-length LcrV antigen (required for secretion of virulence effectors proteins and a virulence factor by itself) from the three independent guaBA, htrA, and rpoS chromosomal sites, each containing an osmotically-regulated P_(ompC)-lcrV cassette. The protective anti-phagocytic capsular F1 antigen was expressed from the SSB-stabilized non-antibiotic low copy expression plasmid pSEC10, creating the plasmid pSL445. The F1 antigen of pSL445 was encoded by the natural Y. pestis caf1 operon but engineered to be transcriptionally controlled by the in vivo-inducible sifA promoter (an S. Typhi promoter controlled by the Salmonella Pathogenicity Island 2 (SPI 2) regulon), after having determined that expression of caf1 using the P_(ompC) promoter was toxic to CVD 910. For comparison, the bivalent plasmid pSL483 (again derived from pSEC10) was also constructed in which the expression of both the caf1 operon and lcrV were divergently transcribed from the P_(sifA) and P_(ompC) promoters respectively. SL483 was then introduced into CVD 908-htrAssb creating a bivalent plague candidate vaccine CVD 908-htrAssb(pSL483) in which foreign antigen expression was completely plasmid encoded, to be compared to CVD 910-3Lssb(pSL445) in which foreign antigen expression was balanced between the chromosome and a plasmid.

The immunogenicity of these live vector vaccine strains was evaluated using a heterologous prime-boost immunization strategy in which BALB/c mice were primed intranasally with 1×10⁹ CFU of live vaccine on days 0 and 28, followed by a boost with a small amount (500 nanograms) of purified lcrV adsorbed to alum on day 56. All mice were challenged on day 84 (i.e., 28 days after the last immunization) with 177 LD50s of virulent Y. pestis strain CO92. Results are presented in Table 6.

TABLE 6 Immunogenicity of S. Typhi live vector candidate plague vaccines expressing LcrV and F1 and further tested for efficacy in a lethal pulmonary challenge model. Day 28 Day 56 Day 84 Percent survival (before (before (4 weeks (14 days Vaccine boost 1) Day 42 boost 2) post boost 2) post challenge) F1-specific serum IgG CVD 910 12.5 12.5 12.5 12.5 40% CVD 910-3L 212.5 12.5 12.5 12.5 70% CVD 910-3Lssb(pSL445) 2,268.3 33,810.9 16,613.2 21,778.3 100%  CVD 908-htrAssb(pSL483) 445.2 11,056.1 1,706.5 3,684.7 100%  PBS prime-LcrV boost 12.5 12.5 12.5 12.5 20% PBS 12.5 12.5 12.5 12.5  0% LcrV-specific serum IgG CVD 910 49.4 125.0 12.5 78,994.1 CVD 910FL 93.9 224.6 12.5 86,968.7 CVD 910-3Lssb(pSL445) 25.0 75.4 12.5 228,230.1 CVD 908-htrAssb(pSL483) 25.0 20,008.9 21,267.3 407,085.8 PBS prime-LcrV boost 25.0 51.3 12.5 28,855.7 PBS 25.0 25.0 12.5 12.5 Typhi LPS-specific serum IgG CVD 910 168.5 3,570.8 Pending 30,192.9 CVD 910-3L 311.8 7,088.0 18,754.0 52,266.0 CVD 910-3Lssb(pSL445) 171.5 1,366.9 Pending 18,917.4 CVD 908-htrAssb(pSL483) 135.3 1,248.5 Pending 1,968.6 PBS prime-LcrV boost 157.8 121.3 307.0 244.2 PBS 150.9 116.5 297.6 188.7

These results clearly show that when expression of foreign antigens is balanced between inducible multilocus chromosomal expression and inducible plasmid-based expression, serum antibody responses against both foreign antigens LcrV and F1 were equivalent to that observed when both antigens were expressed from a single stabilized expression plasmid. Perhaps more importantly, when examining live vector-specific LPS responses, serum IgG responses 10 fold higher were observed in mice immunized with CVD 910-3Lssb(pSL445) versus responses in mice immunized with CVD 908-htrAssb(pSL483) (day 84 GMT=18,917.4 versus 1,968.6 respectively). These results strongly support the hypothesis that the metabolic burden associated with expression of multiple foreign antigens in attenuated multivalent live vector vaccines can be reduced or even eliminated by engineering appropriately balanced levels of antigen expression, accomplished by strategic distribution of foreign genes between multiple chromosomal loci and genetically stabilized low copy plasmids.

Construction of a Multivalent CVD 910 Live Vector Vaccine Targeting Enterotoxins and a Putative Colonization Factor of C. difficile.

Enterotoxins A (TcdA) and B (TcdB) are the primary virulence factors of C. difficile. However, epidemic strains of C. difficile invariably carry an additional toxin affecting the actin cytoskeleton of intestinal cells called C. difficile transferase (Cdt); this toxin has also been called binary toxin (BT) because it is composed of a catalytic A subunit and a cell-binding B subunit (30, 31). The activity of BT causes rearrangement of the actin cytoskeleton of intestinal epithelial cells, disrupting tight junctions and potentially allowing better penetration of enterotoxins into gastrointestinal tissue (32), thereby enhancing the virulence of epidemic strains. It was recently discovered that BT also appears to enhance colonization of the intestinal tract by inducing microtubule-based protrusions which enhance the adherence of C. difficile (33). Based on available data, it was hypothesized that binary toxin acts to enhance the virulence of epidemic strains carrying all three toxins by promoting better colonization of C. difficile and possibly improving the penetration and binding of enterotoxins A and B. Recent animal studies suggest that immunization against enterotoxins alone does not prevent colonization of the gastrointestinal tract by C. difficile (34), and that strains producing only binary toxin are able to colonize susceptible animals (35). Therefore, live vector-mediated mucosal immunity against C. difficile disease will be targeted at three levels: 1] by blocking the binding of both enterotoxins through targeting of serum immunity to their cell-binding domains, 2] by inducing mucosal immunity to the cell binding domain of BT (designated here as CBD-BT) to reduce penetration of toxins A and B by maintaining the integrity of intestinal epithelial tissue, and 3] by targeting mucosal immunity against CBD-BT to reduce intestinal colonization, recurrent infection, and environmental transmission in a clinical setting.

Towards this goal of constructing a trivalent live vaccine against C. difficile infections in which mucosal immunity is targeted against enterotoxins A, B, and binary toxin, CVD 910-3A was first modified to express CBD/A from one further chromosomal locus, namely the clyA locus. This modification serves to avoid loss of expression of chromosomally encoded 14CBD/A from the three chromosomal loci of CVD 910-3A. Integration of P_(ompC)-14cbd/a into the clyA locus was completed using the method described above in the paragraph entitled “Construction and testing of CVD 910-3A”; this integration cassette was integrated such that only the open reading frame encoded by clyA was replaced, while preserving the original clyA promoter, as depicted schematically in the chromosomal integration strategy of FIG. 3.

The resulting monovalent vaccine strain, CVD 910-4A, was then further modified to contain and express CBD-BT fused to the carboxyl terminus of ClyA in the manner routinely used in low copy number SSB-stabilized expression plasmids; as with other expression cassettes constructed in these plasmids and later moved into chromosomal integration modules, this clyA::cbd-bt gene fusion was again transcriptionally controlled by the osmotically regulated promoter P_(ompC). In order to take advantage of the previously constructed integration modules in which incoming expression cassettes encoding foreign antigens were inserted as EcoRI-AvrII fragments, it was necessary to create a new synthetic gene encoding ClyA in which the internal naturally occurring EcoRI site was removed; the resulting synthetic gene (designated clyA*) was then re-inserted into pSEC10 to create pSEC10S2 (SEQ ID NO:45). In addition to the EcoRI site (base 1034, T to C), other commonly used restriction sites in pSEC10S2 (SEQ ID NO:45) were removed, including BglII (base 590, T to C), two HindIII sites (base 686, A to G; base 1043, A to G), HpaI (base 978, T to C; base 980, A to G) and MfeI (base 1262, A to G).

A synthetic 2655 bp codon-optimized gene encoding the 878 residues of CBD-BT (SEQ ID NO:46) was then synthesized as a NheI-AwII fragment (SEQ ID NO:47). However, insertion of this cassette into pSEC10S2 cleaved with NheI was unsuccessful. A smaller gene cassette encoding residues 212-878 of CBD-BT (designated B2; SEQ ID NO:46) was amplified using the forward primer 5′-AGATCTaaaataaggaggaaaaaaaaATGGCTAGCCTGATGTCTGATTGGGAAGATGAAG-3′ (NheI site underlined; SEQ ID NO:51) and the reverse primer 5-AAGCTTCCTAGGTTATTAATCCACACTCAGAACCAGCAGTTCG-3′ (AvrII site underlined; SEQ ID NO:52); this decision was based on a report by Sundriyal et al. (36) demonstrating the biological activity of this truncated portion of CBD-BT which results naturally from proteolytic cleavage of the full-length 98.8 kDa CBD-BT, removing a 20 kDa N-terminal fragment, and resulting in a soluble 74.9 kDa protein. The resulting 2054 bp synthetic gene (SEQ ID NO:48) was then successfully inserted as a NheI-AvrII fragment into pSEC10S2 cleaved with NheI to create pSEC10S2-B2 encoding a 972 residue 108.6 kDa ClyA-CBD-BT fusion protein (SEQ ID NO:49). This plasmid was then introduced into CVD 910-4A to create the bivalent vaccine CVD 910-4Assb(pSEC10S2-B2).

The P_(ompC)-clyA*-b2 expression cassette was then excised from pSEC10S2-B2 as a 3491 bp EcoRI-AvrII fragment (SEQ ID NO:50), inserted into the guaBA integration module cleaved with MfeI-NheI, and then integrated into the guaBA locus of CVD 910-4A, replacing the previously integrated P_(ompC)-14cbd/a cassette to create the bivalent live vaccine CVD 910-3A-GB2 in which 14CBD/A is chromosomally expressed from the htrA, rpoS, and clyA loci, and CBD-BT is expressed from the guaBA locus. When export of the protein fusion was compared for hemolytic activity with plasmid-encoded fusion protein expressed in CVD 910-4Assb(pSEC10S2-B2), proper export of the fusion protein was observed in both strains, although plasmid-encoded export was much higher due to copy number (FIG. 6).

The SSB-stabilized plasmid encoding the cell binding domain of enterotoxin B, pSEC10-CBD/B, is introduced into CVD 910-3A-GB2 to create the trivalent live vaccine CVD 910-3A-GB2ssb(pSEC10-CBD/B) shown in FIG. 7 in which 14CBD/A is chromosomally expressed from the htrA, rpoS, and clyA loci, CBD-B2 is expressed from the guaBA locus, and CBD/B is expressed from pSEC10-CBD/B.

Alternatively, the SSB-stabilized plasmid encoding the cell binding domain of enterotoxin B, pSEC10-CBD/B, is introduced into CVD 910-2A-GRB2 to create the trivalent live vaccine CVD 910-2A-GRB2ssb(pSEC10-CBD/B) in which 14CBD/A is chromosomally expressed from the htrA and clyA loci, CBD-B2 is expressed from the guaBA locus and rpoS locus, and CBD/B is expressed from pSEC10-CBD/B.

To improve binary toxin-specific toxin neutralizing activity, a second copy of P_(ompC)-clyA*-b2 can be inserted into the rpoS locus of CVD 910-3A-GB2ssb(pSEC10-CBD/B).

While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.

REFERENCES

All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. Each cited patent and publication is incorporated herein by reference in its entirety. All of the following references have been cited in this application:

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What is claimed is:
 1. An attenuated strain of Salmonella enterica serovar Typhi (“S. Typhi”) having chromosomal disruptions in the guaBA locus, the htrA locus, the rpoS locus, and the ssb locus.
 2. The attenuated strain of claim 1, wherein the attenuated strain further comprises a chromosomal-based expression system integrated into at least one location of chromosomal disruption, wherein each chromosomal-based expression system comprises an expression cassette encoding an antigen.
 3. The attenuated strain of claim 2, wherein the attenuated strain comprises at least two chromosomal-based expression systems.
 4. The attenuated strain of claim 3, wherein the attenuated strain comprises two different chromosomal-based expression systems.
 5. The attenuated strain of claim 2, wherein the attenuated strain comprises at least three chromosomal-based expression systems.
 6. The attenuated strain of claim 5, wherein the attenuated strain comprises three different chromosomal-based expression systems.
 7. The attenuated strain of claim 2, wherein the attenuated strain comprises at least four chromosomal-based expression systems.
 8. The attenuated strain of claim 7, wherein the attenuated strain comprises four different chromosomal-based expression systems.
 9. The attenuated strain of claim 2, wherein the attenuated strain further comprises one or more plasmid-based expression systems, and wherein each plasmid-based expression system comprises an expression cassette encoding an antigen.
 10. The attenuated strain of claim 9, wherein the plasmid-based expression system is an SSB-stabilized plasmid-based expression system.
 11. The attenuated strain of claim 2 formulated as a pharmaceutical composition comprising a pharmaceutically-acceptable carrier or diluent.
 12. The attenuated strain of claim 9 formulated as a pharmaceutical composition comprising a pharmaceutically-acceptable carrier or diluent.
 13. The attenuated strain of claim 1, wherein the attenuated strain further comprises (i) a chromosomal-based expression system integrated into at least one location of chromosomal disruption, wherein each chromosomal-based expression system comprises an expression cassette encoding an antigen; and (ii) one or more plasmid-based expression systems, wherein each plasmid-based expression system comprises an expression cassette encoding an antigen.
 14. The attenuated strain of claim 13 formulated as a pharmaceutical composition comprising a pharmaceutically-acceptable carrier or diluent. 